Method of preparing autologous cells and methods of use for therapy

ABSTRACT

A method for expanding mesenchymal cells derived from autologous bone marrow in autologous culture medium which can be used in a clinical setting, and a business method for performing such expansions in the future as a service for patients. A method for expanding mesenchymal cells derived from autologous bone marrow in autologous culture medium including a diagnostic kit for the autologous cell therapy to determine whether a patient will respond to the autologous cell therapy for treatment of a disease, in which said kit comprising a system for detecting gene and protein expression comprising at least two isolated DNA molecules wherein each isolated DNA molecule detects expression of a gene that is differentially expressed in the tissue of the patient that is intended to be the source of the autologous cell therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/434,537, filed May 1, 2009, now U.S. Pat. No. 9,301,975, the entirecontent of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 18, 2016, isnamed 29181-709.301_SL.txt and is 1,585,972 bytes in size.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The inventions described below relate the field of autologous celltherapy.

In our prior U.S. patent application Ser. No. 11/735,869, filed Apr. 16,2007, we described a treatment for patients suffering from chronicmyocardial infarction by injecting autologous bone marrow cells in ornear the areas of chronic myocardial infarction in the patient's heart.In addition to the mononuclear cells, mesenchymal cells, CD-34 positivecells, CD-90 positive cells, and CD-133 positive cells mentioned intherein, additional cell therapies may be applicable to other cardiacconditions. Each of these cell therapies may be useful to treat chronicmyocardial infarction, acute myocardial infarction, myocardial ischemia,chronic myocardial ischemia, heart failure, cardiovascular disease, andperipheral vascular disease. U.S. patent application Ser. No.11/735,869, U.S. is hereby incorporated by reference.

Human bone marrow mesenchymal cells (hMSCs) are currently beinginvestigated for a number of clinical applications includingcardiovascular repair, orthopaedic repair, connective tissue repair, andimmune diseases such as graft versus host disease, Crohn's disease. Theyhave potential roles in other immune diseases such as lupus,osteoarthritis and rheumatoid arthritis as well as well as diabetes.They also have potential to act as carriers of gene-based therapeuticswith ex vivo transfection of the cells to enhance the control of genebased therapeutics dosing. The following prior art references are herebyincorporated by reference: U.S. Pat. Nos. 7,101,704; 7,029,666;6,875,430; 6,863,900; 6,835,377; 6,797,269; 6,761,887; 6,709,864;6,685,936; 6,541,024; 6,387,369; 6,387,367; 6,379,953; 6,368,636;6,358,702; 6,355,239; 6,342,370; 6,328,960; 6,322,784; 6,281,012;6,261,549.

Although it has been argued that allogenic cells have great potential toprovide an off the shelf product for patients because they will not berecognized as foreign by the recipient patient, this has not yet beenproven. Allogenic cells have a greater propensity (compared toautologous cells) to carry diseases from the donor to the recipient andhave a greater propensity for rejection of the therapeutic cells by theimmune system of the patient who received them.

Expansion of autologous hMSC in a truly autologous culture medium thatcontains no animal serum or allogenic culture additives for clinicaltransplantation to the patient, which has not previously been proposed,should also be valuable avoiding the problems associated with allogeniccells. To date, reported clinical trials are employing human bone marrowmesenchymal stromal cells generated in a culture medium supplementedwith fetal calf serum (FCS). FCS is an undesired source of xenogeneicantigens and bears the risk of transmitting animal viral prion andzoonose contaminations. Additionally FCS has been implicated withanaphylactic or arthus like immune reactions in patients who receivedcells generated in FCS supplemented medium even leading to arrhythmiasafter cardioplasty (Chachques 2004).

Early work on autologous serum hMSC has recently been shown to bepromising by Stute et al 2004, but the amount of autologous serumrequired is identified as a significant hurdle to clinical expansionsuch that the investigators proposed using 1% and 3% autologous serum inthe culture medium even though they had far superior results at 10%autologous serum. Other investigators have also shown a fasterproliferation compared to FCS at least during first passages withoutloss of the typical phenotype, motility, and differentiationcapabilities in vitro, whereas allogenic human serum resulted in hMSCgrowth arrest and death (Kobayashi et al 2005, Shandahar et al 2005,Mizuno et al 2006).

The primary reason presented that autologous serum cannot be consideredas a general substitute for FCS is the amount of autologous serumnecessary for sufficient expansion would exceed the amount a donor couldprovide (Lange et al 2007). Lange et al 2007 proposed the use of pooledplatelet lysates to create an activated plasma to avoid the use of FCS,but their solution falls short of that provided here in that they arepooling the platelets from multiple donors to create an allogenicculture medium.

Here we disclose three methods to culture bone marrow or adipose tissuederived autologous culture medium expanded autologous mesenchymal stemcells (ACMEAM's) from the bone marrow or adipose tissue. These inventivemethods have enormous value and have been validated in both a swine andhuman and are comparable to the gold standard of culturing in 10% fetalcalf serum.

BRIEF SUMMARY OF THE INVENTION

The methods and devices described below provide for treatment of severalcardiovascular diseases with autologous culture medium expandedautologous mesenchymal stem cells (ACMEAM's) from the bone marrow. Totreat a patient with the various cardiovascular diseases, includingchronic myocardial infarction, myocardial ischemia, acute myocardialinfarction, congestive heart failure, atherosclerosis, coronary arterydisease, and peripheral artery disease, blood components and a source ofMSCs (typically bone marrow or adipose tissue stromal cells) areextracted from the patient. Autologous culture medium is prepared fromthe blood components and the extracted MSCs are expanded to createACMEAMs over a period of weeks for subsequent characterization andadministration to the patient. The cells are expanded in the autologousculture medium developed from the blood components as described. Whensufficient population of expanded cells has been grown, the cells areadministered to the patient from whom they were harvested, in or nearthe site of the disease to be treated.

These therapies are extremely expensive, perhaps $10,000 to $50,000 pertherapeutic dose of approximately 20 to 100 million cells delivered in avolume of 1.0 to 5.0 ml which is typically, but not necessarily, used ina single session of tissue injections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method for preparing autologous culture mediumexpanded autologous mesenchymal stem cells (ACMEAMs) from the bonemarrow.

FIGS. 2a through 2d show comparative photographs of Swine mesenchymalstromal stem cells taken from the bone marrow and cultured for 18 daysin (FIG. 2a ) 10% fetal calf serum, (FIG. 2b ) 10% autologous serum,(FIG. 2c ) 5% platelet rich plasma where platelets were lysed with afreeze thaw cycle, and (FIG. 2d ) 5% platelet rich plasma whereplatelets were lysed with a thrombin activation.

FIGS. 3a through 3d show comparative photographs of Human mesenchymalstromal stem cells taken from the bone marrow and cultured for 11 or 13days in (FIG. 3a ) 10% fetal calf serum, (FIG. 3b ) 10% autologousserum, (FIG. 3) 5% platelet rich plasma where platelets were lysed witha freeze thaw cycle, and (FIG. 3d ) 5% platelet rich plasma whereplatelets were lysed with a thrombin activation.

FIG. 4 is a table of CD Profiles in Human Cultured Autologous BoneMarrow Mononuclear Cells in Fetal Calf Serum (FC), Autologous Serum(AS), Freeze and Thaw Platelet Activated Plasma, (F&T PRP) and ThrombinActivated Platelet Activated Plasma (tPRP).

DETAILED DESCRIPTION OF THE INVENTIONS

Three separate preferred methods for creating autologous culture mediumare provided. These involve adding to a base medium one of the followingthree (1) autologous serum at 10% by volume, (2) activated plasmadeveloped from thaw freeze platelet lysates at 5% by volume, and (3)activated plasma made by thrombin activation at 5% by volume.

(1) ACMEAMs in Autologous Serum

The treatment method starts with harvesting both blood from the patient,and harvesting stem cells from the patient. It has been noted in theliterature that the generation of autologous serum for culturing isdifficult in that many patients cannot provide sufficient blood toenable expansion of the stem cells from the patient in the serum fromthe patient. The solution here on the surface will appear quite simple,but it is not the preferred embodiment of this invention as it is farmore complex clinically than the other two solutions.

The problem that makes autologous serum not a solution for patienttherapeutics is that many patients, particularly those with asignificant cardiovascular disease, cannot donate sufficient blood in asingle visit to enable successful cell growth. This problem of obtainingenough autologous serum in a single visit to culture cells, is solved byhaving them donate the tissue for culturing on multiple separateoccasions with a sufficient time lag between the blood harvest for themto have regenerated. In this invention, the patient's own serum will beused to avoid fetal calf serum and provide for autologous serum to growmesenchymal cells taken from bone marrow. To obtain this serum, 250 mlof blood will be taken from each patient at each of two visits separatedbetween one and three weeks apart for a total blood volume of 500 ml ofblood. This blood will be stored as a source for autologous tissueculture medium. This type of thinking goes against typical medicalpractice as one desires to have the patient see the physician as littleas possible, and spend as little time as possible within the hospital.Physician and hospital time is expensive. The additional time togenerate the autologous serum also slows the delivery of therapy whichis also not desirable. Further, having multiple tissue harvest pointsrequires additional control in tracking the blood volumes that will beused to form serum as well as the cell source from which the mesenchymalcells will be expanded. However donating blood products at two or moretime points before the therapy does provide a solution to generatingautologous serum.

The disadvantage of the delay in cell processing could be addressed bycontinuing to harvest tissue during the culturing process as the needfor autologous serum increases exponentially over time with theexpansion of the cultures. Although this is theoretically feasible, itmakes no sense from a manufacturing process perspective as many logisticissues could result in a lack of inputs at critical periods during theculture process.

FIG. 1 shows this method and neatly solves the hurdle of both enablingthe development of autologous serum to expand ACMEAMs as well as a fallback strategy to deliver a preparation of MNCs should the ACMEAMs notculture as expected. Patients will have up to 150 ml of bone marrowaspirated under local anesthesia. Up to 100 ml will be used to expandthe mesenchymal cell population in this autologous serum to develop adosage of up to 100 million MSCs, with a minimum of 20 million MSCs.Patients who are not eligible for MSCs due to difficulties in culturinga minimum of 20 million MSCs may be eligible to receive frozen MNCs at adosage of 100 million cells which is the same dosage previouslydelivered by our group in a clinical setting with good results.

In this invention, 250 ml of blood will be taken from the patient toobtain autologous serum roughly five weeks before therapy is scheduledand an additional 250 cc will be taken roughly four weeks before therapyto provide for 500 cc of blood to provide for autologous serum. Roughlythree weeks before therapy (such as cardiac catheterization for deliveryin a setting of heart failure) 150 ml of autologous bone marrowmononuclear cells will be aspirated under local anesthesia from theposterior iliac crest. Bone marrow mononuclear cells will be isolated bydensity gradient on Ficoll-Paque Plus tubes (Amersham Biosciences).Cells will be washed and filtered through 100 .mu.m nylon mesh to removecell aggregates.

The blood and bone marrow are transported to a laboratory forprocessing, including separation of the mesenchymal stem cells from thebone marrow, and culturing and expanding a portion of mesenchymal stemcells (while preserving a portion of the mesenchymal stem cells forlater use, including re-supply of mesenchymal stem cells should theprocessed portion fail). The mesenchymal stem cells are expanded until apopulation of up to 200 million cells is achieved.

Cells are divided into two fractions. One fraction will be cultured inthree (3) T225 cm.sup.2 Falcon flasks (Becton Dickinson) providing cellsthat will then be seeded into thirty (30) T225 cm.sup.2 flasks togenerate up to 200 million P1 cells using the autologous serum. Tenpercent of the final cell solution will be used for cell counting andviability testing using trypan blue exclusion and bacteriologicalanalysis. A second fraction will be frozen for future use if the numberof cultured hMSCs do not reach the acceptable 20 million level. Thissecond fraction may be used in an attempt to deliver MNCs as shown inFIG. 1 or saved to attempt an expansion of the frozen cells at a laterdate.

The expanded population of up to 20M cells is then frozen andtransported to the medical facility where the treatment is to beperformed, and cells are thawed, washed, and re-suspended just prior todelivery at a concentration of up to 4.0.times.107 cells/ml in a totalvolume of up to 5 ml.

(2) ACMEAMs in Activated Plasma with Platelet Freeze Thaw

Blood components (serum, plasma, platelets and their derivatives) areobtained through apheresis according to standard procedure. There arenumerous types of apheresis. Blood taken from a healthy donor can beseparated into its component parts, where the needed component iscollected and the “unused” components are returned to the donor. Fluidreplacement is usually not needed in these type of collections. Thereare large categories of component collections: Plasmapheresis—bloodplasma. Plasmapheresis is useful in collecting FFP (fresh frozenplasma). Plateletpheresis (thrombapheresis, thrombocytapheresis)—bloodplatelets. Plateletpheresis, like it sounds, is the collection ofplatelets by apheresis; while returning the red blood cells, white bloodcells, and component plasma. The yield is normally the equivalent ofbetween six and ten random platelet concentrates.Leukapheresis—leukocytes (white blood cells). Leukopheresis is theremoval of PMN's, basophils, eosinophils typically for transfusion intopatients whose PMN's are ineffective or traditional therapy has failed.

Apparatus such as Haemonetics MCS+9000 or COBE 2991 with sterile singleuse consumables are used. After the venous access is secured, the speedis set up to 60 ml/min. A maximum of 15% of the total blood volume issampled according to the standard tables (i.e. a 75 KG patient has atotal blood volume of 5250 ml and 787 ml may be sampled). The red cellsare infused back. The whole procedure takes 1-2 hours and yields10.sup.11 to 5.times.10.sup.11 platelets.

The Apheresis produces platelet rich plasma (up to 250 ml) which aresplit into 50 ml bags using sterile techniques. Bone marrow is processedas before on the same day and sent to the processing lab in the samepackage with the platelet rich plasma.

At the processing lab, the platelet rich plasma bags are then frozen inliquid nitrogen for 5 seconds and then thawed under 37 .degree. C. waterand then frozen again in liquid nitrogen. After the second thaw they aretransferred into 15 ml falcon conical tubes and frozen at −20 .degree.C. These tubes are then thawed for the culture medium and added to theculture base medium alpha MEM from GIBCO at 5% to 10% upon which themononuclear cells containing mesenchymal cells obtained as describedpreviously are plated out similarly as previously described.

The advantage of this approach is that even with a large marrowaspiration of 150 ml, the total volume loss for the patient is only 150ml. This can be done on volumes of marrow as small as 50 ml. Further,the procedure for quality control just became enormously simpler as thepatient presents at the hospital at one time point and the reagents forcell culture are shipped and processed at the lab at one time point.This is enormously valuable.

(3) ACMEAMs in Activated Plasma with Platelet Thrombin Activation

Platelet rich plasma and autologous bone marrow are obtained and shippedto processing lab on the same day as described previously.

At the processing lab, one unit of thrombin per ml is added to theplatelet rich plasma bags containing 150 ml. Thrombin activated plateletrich plasma is then gently shaken for 45 minutes at room temperature,followed by transfer into 50 ml conical tubes and spun to eliminateplatelet aggregates. Supernatent is aliquoted in 15 ml tubes and frozenfor further supplementing the culture medium. These tubes are thenthawed for the culture medium and added to the culture base medium alphaMEM from GIBCO at 5% to 10% upon which the mononuclear cells containingmesenchymal cells obtained as described previously are plated outsimilarly as previously described. This has the same advantages of thefreeze thaw platelet lysate autologous culture medium.

FIGS. 2a through 2d show cultured swine MSCs 18 days after culture withthree autologous mediums invented here. Swine mesenchymal stromal stemcells taken from the bone marrow and cultured for 18 days in (FIG. 2a )10% fetal calf serum, (FIG. 2b ) 10% autologous serum, (FIG. 2c ) 5%platelet rich plasma where platelets were lysed with a freeze thawcycle, and (FIG. 2d ) 5% platelet rich plasma where platelets were lysedwith a thrombin activation. They show very similar characteristic toclassic MSC morphology.

FIG. 3a through 3d show human mesenchymal stromal stem cells taken fromthe bone marrow and cultured for 11 or 13 days in (FIG. 3a ) 10% fetalcalf serum, (FIG. 3b ) 10% autologous serum, (FIG. 3c ) 5% platelet richplasma where platelets were lysed with a freeze thaw cycle, and (FIG. 3d) 5% platelet rich plasma where platelets were lysed with a thrombinactivation. They show very similar characteristic re MSC morphology. Allcultures remained viable and non-contaminated until the end (4 weeks ofculture). Cellularity was good with fetal calf serum and platelet richplasma with freeze thaw but less optimal with autologous serum orthrombin activation.

FIG. 4 shows the flow cytometry CD Profiles in Human Cultured AutologousBone Marrow Mononuclear Cells in Fetal Calf Serum (FC), Autologous Serum(AS), Freeze and Thaw Platelet Activated Plasma, (F&T PRP) and ThrombinActivated Platelet Activated Plasma (tPRP). Profile of cells from the 4different culture conditions showed a similar phenotype: CD105+, 73+,90+, 34−/45−−. This experiment on all patients had essentially identicalresults. The only anomalies are shown in gray in the first patient run.The Row denoted “expected in” shows expected percentage, and on whattype of cells the CD is known to be expressed. The five columns with a *are the 5 CDs sufficient to prove that the cells are a form of MSCs.

For therapeutic purposes the cells are then frozen, shipped to theinterventional lab and delivered to the patient. For cardiacapplications the preferred deliver is transendocardial intramyocardialdelivery or surgical direct injection into the target zone. Forgastroesophogeal reflux the cells are delivered through a catheter ortransesophogeal ultrasound probe having a penetrating needle elementinto the region of weakening at the stomach-esophagus junction. ForCrohn's disease they are delivered via the rectum through agastrointestinal catheter designed to navigate in this anatomy with asimilar penetrating element and distal steerable member.

The cell compositions and suitable delivery catheters can be provided ina combination cell and device therapeutic. A disposable single usecatheter having a distal penetrating needle tip for insertion intotarget tissues, with the distal penetrating element in fluidcommunication with a fluid port on the proximal end of said catheter maybe used for delivery. Using such a catheter, the cell composition isinjected interstitially into the tissue. The cells are delivered at aconcentration of between 20M and 40M cells per cc at a dosage of between20M and 200M cells total. The catheter and a reservoir of cells may bepackaged at a manufacturing site and shipped as an assembly to a medicalfacility for immediate use.

These and other autologous therapeutic cells all have diagnostic valueas well. Because these cells are autologous they hold information aboutthe therapeutic potential of the cell and also about the characteristicsof the patient that the cultured cells will be administered to that mayreflect their responsiveness to therapy. Central to moleculardiagnostics is the tissue that is assayed for biomarkers. Throughoutthis disclosure, the preferred embodiment of tissue for analysis isblood as it is readily available in all patients at all times, and itcarries within it the cells of the immune system which recognize knownand as yet unknown diseases. Blood is occasionally the source for cellbased therapies, and is in intimate communication with other sourcesfrom which cell based therapies may be developed and derived: the bonemarrow, the adipose tissue, the heart tissue, the nervous systemtissues, the skin, the pancreas, etc. However in this specification, itwill be recognized that the cells that are the specific source of thecell therapy will have even greater potential to be characterized fortheir potency in addition to the patient responsiveness to such atherapy due to genetic makeup.

In addition to the mesenchymal stem cells described here, all autologouscell therapy preparations may benefit from this diagnostic strategy.This includes the autologous bone marrow derived cells and cellpopulations in development at Aldagen and Aastrom Biosciences, as wellas the CD34+ cells derived from blood in development at BaxterHealthcare, the autologous cells derived from Adipose tissue indevelopment by Cytori Therapeutics, and other similar cell basedtherapeutics. Being able to characterize cells for potency by measuringtheir gene expression provides a means to avoid expensive and riskyinterventions for those who are less likely to respond. It also providesa means to optimize and enhance this therapeutic strategy over time. Forexample, if patients without expression of RNAs that code for angiogeniccytokines is noted, these patients may then be selected to receive amodified therapy that includes an angiogenic cytokine—such as describedin the trial described by Losordo in which cells are transfected toexpress

VEGF prior to delivery (ClinicalTrials.gov Identifier: NCT00279539).

Example 1

In a first blood draw before tissue is harvested for therapeuticpurposes, the gene expression or proteomic patterns in the circulatingblood leukocytes are used to determine whether (and to what extent) thepatient will respond to various potential cell therapy preparations. Inthis setting there are options for the preparation of tissue foranalysis including: PreAnalytix Pax Gene tubes which are commerciallyavailable to stabilize whole blood RNA, isolation of the leukocytesusing gravity centrifugation methods and then preserving these witheither a PaxGene tube or freezing these cells on dry ice and ultimatelyat −80 C, or taking fractions of the blood using flow cytometry toremove subsets of cells using antibody probes for surface markers ofinterest which may be more relevant to the therapeutic strategy inquestion and ultimately provide for a greater signal to noise ratio whenlooking at differences between patients as the noise of other cell typeswill not be present. Further, the blood sample may include the serum andeven the RBCs which may be used in proteomic and other analysis usingvarious cytokine protein microarrays and mass spectrometrymethodologies.

In its most likely first embodiment the gene expression or proteomicassay will enable a physician to determine whether or not a patient islikely to be responsive to their cells for the clinical indicationselected. The gene expression or proteomic patterns may indicate that(1) a simple autologous bone marrow mononuclear cell (ABM MNC)preparation, (2) a preparation of autologous bone marrow mesenchymalcells, (3) an unmodified adipose-derived stromal cell population, or (4)an adipose-derived autologous serum expansion of mesenchymal cells isbest suited to treat the patient. This has not been performedpreviously. The gene expression or proteomic patterns can be detectedwith assays such as pre-existing assays whole genome microarraysprovided by Agilent or Affymetrix and then identifying high fold changegenes and using bio-informatic techniques to build classifiers whichenables a smaller set of genes to be assayed using PCR or other highresolution measurement approach on a smaller set of genes (.about.20 orso). The methods for analyzing gene expression data included principalcomponents analysis, linear discriminant analysis (LDA, StatSoft, Inc.),logistic regression (SAS Institute, Inc.), prediction analysis ofmicroarrays (PAM) voting, classification and regression trees (TreeNet,Salford Systems), Random Forests, nearest shrunken centroids andk-nearest neighbors.

Once a small set of genes is selected confirming its potential remainsnontrivial. Some groups (Deng et al American Journal of Transplantation2006; 6: 150-160) have used these techniques to develop a lineardiscriminant analysis system which is relatively simple, otherapproaches are also possible which do not reduce the output to a simplescalar score. One approach involves ranking the genes for their abilityto independently separate the groups and constructing a map thatconsiders the independent score of each gene coupled with the relativescore based on all more significant genes. Each gene creates threescenarios: A probability greater than 50+X % that patient is aresponder, B probability greater than 50+X % that patient is anon-responder, or AB indeterminant in which neither A nor B is true. Xmay be selected and need not be that large for this technique to workwell. The first gene then defines three classes: A1, B1, and AB1. Thesecond gene builds on this scoring and one gets nine separateclassifications possible: A1A2, A1B2, A1AB2, B1A2, B1B2, B1AB2, AB1A2,AB1B2, AB1AB2. A third gene similarly results in 81 independent statesthat may be assessed and so on. Such a graphical readout is possible fortens of genes which with color coding of the As and Bs one can begin toreadily interpret patterns where there is high confidence on therapeuticeffectiveness without discarding important data that may be useful forfuture analysis and interpreted graphically for up to 20 ranked genes(including quality control genes such as plant genes). If X is selectedto be vanishingly small, a binary score is possible that makes trackingthe decision aspects of each gene contributor to the algorithm muchsimpler.

As yet undefined algorithms may be employed in this invention as thereis significant ongoing work in this area.

The tissue source for therapeutic cells can also be used for diagnosticpurposes. In the preferred embodiment autologous bone marrow mononuclearcells (MNCs) are obtained in a small aspirate and sent to the laboratoryto assess (1) potency of ABM as a therapeutic for a clinical indication,(2) the relative potency of the mesenchymal stromal cells that would becultured from these cells, and (3) the ease and or likelihood that MSCsmay be successfully cultured from the MNCs.

All autologous cell preparations may benefit from this strategy. Furtherthese techniques may be applied to a wide host of clinical situationssuch as Graft Versus Host disease, Crohn's disease and other autoimmunediseases, periodontal repair, orthopaedic repair, and the like.

(4) Determining Susceptibility to Treatment

The following describes a method to determine, roughly, thesusceptibility of patient populations to the autologous cell therapy.This description could address many different diseases such as heartfailure, acute myocardial infarction, chronic ischemia, cardiovasculardisease, peripheral vascular disease, or other cardiovascular disease aswell as Crohn's disease and other autoimmune diseases, periodontalrepair, and orthopaedic repair. For clarity we shall focus on heartfailure. To accomplish this, small populations of patients sufferingfrom heart failure are tested both for a wide array of gene expressionsand for response to cell therapy. These small populations of patientsare tested for a large number of gene expression signatures, markersetc., which may be detected with whole genome assays and interrogatingnumerous variables in the genome analysis. These patients are alsotreated with suitably promising autologous cell therapies, such asACMEAMs (autologous serum expanded autologous mesenchymal cells), wherethe cells are injected into diseased cardiac tissue of the patient.After this therapeutic injection, patients are tested for typicalindications of cardiac health, such as cardiac output, ejectionfraction, ventricular wall motion, exercise tolerance, quality of lifeand the like (including many advanced invasive and non-invasivemeasurements).

Patients are then divided into groups, essentially those that areresponsive to the treatment and those that are not. Each group is testedwith a whole genome assay, which is interrogated under many variables ingenomic analysis. The genomic assays of the two groups are compared, toascertain differences in gene expressions that are consistent betweenthe two groups. From the comparison of the gene expressions fold changesfound in the responsive population to those found in the non-responsivepopulation, candidate genes which are expressing differentially betweenthe two groups. The goal is to develop a set of up to 20 genes that maybe measured from the same autologous tissue (preferably from the sourcematerial of the therapeutic cells) indicative of a responder, but toidentify a pattern of gene expressions that are indicative of aresponder. Numerous genes may express identically in both populations,but several genes will express differentially. Those genes may beignored, and need not be assayed, so that the assay consists of test forthose genes which express differentially between responding andnon-responding groups. Thus, the gene expression/proteomic signatureprofile to determine responders, or to determine non-responders, isdeveloped with a small group of patients. Such an assay is bestvalidated on a second independent population in a subsequent clinicaltrial.

Genes of particular interest might include genes listed SEQ ID NO: 1through SEQ ID NO: 8832 of U.S. Pub. 20070037144 incorporated herein byreference.

After determining the group of gene expressions that indicateresponsiveness (or that indicate non-responsiveness) to a particularcell therapy for a particular cardiac or cardiovascular condition,assays may be manufactured using RT-PCR or other similar technologies todetect the gene expressions indicative of responsiveness, ornon-responsiveness, to one or more potential cell therapies. Prospectivepatients are then tested with the assays to determine if they are likelyto be responsive, or non-responsive, to a particular cell therapy. Thisis accomplished by testing the cells obtained from the patient,subjecting the cells to the assay, and determining from the assay if thepatient is likely to be a responder or a non-responder. It is likelythat in some cases, no information will be available from the assay toinform the effectiveness of therapy.

For those identified as probably non-responsive, cell therapy may beavoided as a likely ineffective treatment, or initiated with a clearunderstanding that it is unlikely to be effective. This would savesignificant expense. For those identified as likely responders, thetherapy may be accomplished by drawing blood in several blood draws,separating the serum from the blood to provide an autologous culturemedium for that particular patient, aspirating a volume of bone marrowfrom the patient, and expanding mesenchymal stem cells derived from thebone marrow in autologous culture medium.

The cell therapy may be targeted on the basis of a single geneexpression if it is clear that the single gene expression is aneffective indicator of a patient's likely responsiveness to theautologous cell therapy. In this case, the method may be implemented byproviding a diagnostic kit to determine whether a patient will respondto an autologous cell therapy for treatment of cardiovascular diseasewhich includes a system for detecting gene expression comprising asingle isolated DNA molecule which detects expression of a gene, asample of autologous tissue the bone marrow, blood, or adipose tissue ofthe patient from which the putative therapeutic is derived and whosegene expression levels are to be measured. Cell therapy may be targetedon the basis of many variable gene expressions, without determination ofa single controlling gene expression, by providing a prognostic kit todetermine whether a patient will respond to an autologous cell therapyfor treatment of cardiovascular disease, which includes a system fordetecting gene expression comprising at least two, and perhaps dozens ofisolated DNA molecules wherein each isolated DNA molecule detectsexpression of a gene. In each instance, the gene expression detected bythe assay is analyzed and compared to the expression of control groupsto determine the susceptibility of the particular patient to the celltherapy. One simple example may be identification of risk factorsrelated to poor angiogenic potency by assessing the cells uponaspiration.

By developing a diagnostic step that involves information on thetherapeutic being delivered in the therapeutic process, additionalinformation on the therapy, its responders, and its non-responders willbe developed over time. Although the initial value is binary decision ofwhether a patient will respond or not because of (i) their geneticmakeup and (ii) the characteristics of the therapeutic autologous cellswill have power over time other details may also emerge regarding bestdosage, best subpopulations of cells, whether or not one or morestrategies to enhance a particular genes expression levels would improvetherapy and the like.

The two value propositions to autologous cell therapies such as thosedescribed here is that they are safe and that they contain informationspecific to the therapeutic and the patient. These value propositionsare believed to be enhanced the closer to truly autologous a putativetherapeutic strategy is.

While the preferred embodiments of the devices, diagnostic kits, andmethods have been described in reference to the environment in whichthey were developed, they are merely illustrative of the principles ofthe inventions. For example, the gene expression signature for a diseasesuch as heart failure could be developed based on surrogate in vitro andin vivo animal assays for potency and subsequently validated and refinedin clinical study. Systemic correlation of in vitro and small animal invivo surrogate potency assays of MCS to gene expression profiles of theharvested source tissue as well as cultured cells from this tissue canbe performed. Potential mechanisms of action of MSCs could be assessedthrough four potency assays. The anti-apoptotic potential of the cellswill be assayed by co-culture with rat myocytes, Hoescht staining of thelater and expression of Bcl2/Bax by western blotting. Growth factorssecretion, by RT-PCR on SDF-1, HGF, IGF and VEGF will be measured asthey are seen as responsible for the pro-angiogenic potential of theMSCs. Cells will also be differentiated in cardiomyocytes using 5azacytidine as transdifferentiation has also been described as aputative mechanism of action. Further surrogate identifiers of potencyfor preclinical development of a gene signature includes: thecolony-forming capacity of BM-MNCs, response of cells to stromalcell-derived factor 1 (SDF-1), response to vascular endothelial growthfactor (VEGF), and in vivo neovascularization capacity as measured bylaser Doppler-derived relative limb blood flow in mice treated withBM-MNCs.

The methods described above provide for development of a population ofautologous stem cells for treatment of disease, by injection of expandedstem cells into tissue of the patient in or near areas affected by thedisease. Development of the population of stems cells is, as describedabove, accomplished by harvesting blood and its components from thepatient, in two or more times separated by a week, and creating aculture medium that is entirely autologous, having no animal serum orallogenic culture additives, but instead being augmented with autologousserum or autologous plasma (activated as described above), and expandingthe autologous stem cells in that culture medium.

Other embodiments and configurations may be devised without departingfrom the spirit of the inventions and the scope of the appended claims.

What is claimed is:
 1. A method of preparing a therapeutic cellpopulation expanded in autologous medium comprising: harvesting bloodfrom a donor in two visits separated between one and three weeks;harvesting bone marrow cells from the donor; forming a culture mediumfrom said blood that contains no animal serum and comprises 10%autologous serum obtained from the donor; and expanding a population ofmesenchymal stem cells from the bone marrow cells in the culture medium.2. The method of claim 1, wherein the population of mesenchymal stemcells is phenotypically characterized by the presence of the followingsurface markers: CD105, CD73 and CD90.
 3. The method of claim 1, whereinthe population of mesenchymal stem cells is phenotypically characterizedby the absence of the following surface markers: CD34 and CD45.
 4. Themethod of claim 1, wherein the population of mesenchymal stem cells isphenotypically characterized by the presence of the following surfacemarkers: CD 105, 73 and CD90, and the absence of CD34 and CD45.